Project Summary Fragile X Mental Retardation Protein (FMRP) is an mRNA-binding translational repressor whose loss causes Fragile X syndrome (FXS), which we fight with a well-established Drosophila model. A revolution in transgenic technology (FlyLight) in this last funding cycle provides unprecedented ability to target individual neurons within brain learning/memory circuitry. We target a single projection neuron (mPN2) with olfactory dendritic input and two synaptic outputs: 1) biased-stereotyped connection in Lateral Horn (innate behavior), 2) activity-dependent probabilistic connection in Mushroom Body (learned behavior). Cross-comparing these synapse classes in one neuron is a phenomenal advantage. We hypothesize simultaneous introduction of sensory activity and FMRP activity-sensor defines the critical period (CP), a restricted developmental time window when early-use activity refines synaptic connectivity and circuit function. In this renewal, we focus on CP activity-dependent translation control in the refinement of synaptic connectivity and circuit function to optimize the mature behavioral output. In Aim I, we test FMRP RNA-binding translational control of critical period structural/functional development at all 3 mPN2 synaptic sites: dendritic input and dual axonal outputs. We use a range of targeted transgenic tools to assay synapse architecture, ultrastructure and molecular assembly, in normal animals compared to the FXS model in staged developmental studies. We introduce targeted optogenetic channels driven throughout the circuit to bidirectionally manipulate activity during CP development, monitoring changes with GCaMP Ca2+ reporters, ArcLight voltage reporters and patch-clamp electrophysiology. In mPN2 developmental studies, we test activity-dependent FMRP roles in CP synaptic connectivity and function. In Aim 2, we test the partnership of FMRP with other RNA-binding proteins (RBPs) in this activity-dependent translation repression mechanism. RBPs rarely act alone, and our preliminary data suggest RBPs Staufen and Pumilio partner with FMRP, with their coupled expression jointly delineating the critical period. We will test formation of FMRP/Staufen/Pumilio complexes and combinatorial roles in mPN2 translation repression. Using double mutant combinations, we will test combinatorial functions in remodeling of CP synaptic connectivity, circuit function and learning/memory behavioral outputs. In Aim 3, we test 3 novel translational targets; 1) phosphatase Corkscrew, 2) ESCRTiii core protein Shrub and 3) Neurobeachin homolog Rugose. Preliminary data show all 3 mRNAs bind FMRP, proteins transiently elevated in the CP in the FXS model and over-expression phenocopies FXS. We hypothesize FXS elevation of 1) Corkscrew increases synaptic signal transduction, 2) Shrub impairs synapse elimination via a local pruning module, and 3) Rugose accelerates synaptic trafficking to impair CP synaptic remodeling. We will test these roles individually with characterized mutants, and test whether CP restoration of protein levels corrects FMRP/Staufen/Pumilio loss phenotypes. FXS is a primary heritable cause of intellectual disability (ID) and autism spectrum disorder (ASD), and targeted interactors are likewise causally linked to ID/ASD states.